This invention relates to a chimeric peptide-nucleic acid fragment, the process for producing the same and its use for appropriately introducing nucleic acids into cell organelles and cells.
It is known that cellular membrane systems are largely impermeable to nucleic acids. However, cell membranes can be overcome very efficiently by physical processes (transformation) and biological processes (infection). Transformation, i.e. the direct absorption of the naked nucleic acid by the cell, is preceded by cell treatment. There are various methods available for the production of these ‘competent cells’. Most processes are based on the observations made by Mandel and Higa (M. Mandel et al., (1970), “Calcium-dependent bacteriophage DNA infection”, J. Mol. Biol. 53: 159-162), who could show for the first time that the yields resulting from the absorption of lambda-DNA by bacteria can be increased fundamentally in the presence of calcium chloride. This method is also used successfully for the first time by Cohen et al. (S. N. Cohen et al. (1972), “Nonchromosomal antibiotic resistance in bacteria: genetic transformation of Escherichia coli by R-factor DNA”, Proc. Natl. Acad. Sci. U.S.A. 69: 2110-2114) for plasmid DNA and was improved by many modifications (M. Dagert et al. (1979), “Prolonged incubation in calcium chloride improves the competence of Escherichia coli cells”, Gene 6: 23-28). Another transformation method is based on the observation that high-frequency alternating fields may break up cell membranes (electroporation). This technique can be used to introduce naked DNA into not only prokaryotic cells but also eukaryotic cell systems (K. Shigekawa et al. (1988), “Electroporation of eukaryotes and prokaryotes: a general approach to the introduction of macromolecules into cells”, Biotechniques 6: 742-751). Two very gentle methods of introducing DNA into eukaryotic cells were developed by Capecchi (M. R. Capecchi (1980), “High efficiency transformation by direct microinjection of DNA into cultured mammalian cells” Cell 22: 479-488) and Klein et al. (T. M. Klein et al. (1987), “High velocity microprojectiles for delivering nucleic acids into living cells”, Nature 327: 70-73): They are based on the direct injection of the DNA into the individual cell (microinjection), on the one hand, and on the bombardment of a cell population with microprojectiles consisting of tungsten, to the surface of which the corresponding nucleic acid was bound (‘shotgun’). The biological infection methods proved their value parallel to the physical transformation of cells. They include particularly the highly efficient viral introduction of nucleic acids into cells (K. L. Berkner (1988), “Development of adenovirus vectors for the expression of heterologous genes”, Biotechniques 6: 616-629; L. K. Miller (1989), “Insect baculoviruses: powerful gene expression vectors”, Bioessays 11: 91-95; B. Moss et al. (1990), “Product review. New mammalian expression vectors”, Nature 348: 91-92) and the liposome mediated lipofection (R. J. Mannino et al. (1988), “Liposome mediated gene transfer”, Biotechniques 6: 682-690; P. L. Felgner et al. (1987), “Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure”, Proc. Natl. Acad. Sci. U.S.A. 84: 7413-7417). All methods described so far deal with the overcoming of the prokaryotic or eukaryotic plasma membrane by naked or packaged nucleic acids. While the site of action is reached already when the nucleic acid are introduced into the prokaryotic cell, further biochemical processes take place in a compartmentalized eukaryotic cell, which support the penetration of the nucleic acid into the nucleus under certain conditions (e.g. viral route of infection in the case of HIV). Analogous infective processes in which exogenous nucleic acids are actively introduced into other cell organelles (e.g. into mitochondria) have not been described so far.
In addition to the introduction of the nucleic acid into the cell and cell organelle, respectively, the transcription and above all the replication of the introduced nucleic acid play a decisive part. In this connection, it is known that the DNA molecules may have a special property which permits duplication in a cell under certain conditions. A special structural element, the origin of the DNA replication (ori, origin), adds thereto. Its presence provides the ability of DNA replication (K. J. Marians (1992), “Prokaryotic DNA replication”, Annu. Rev. Biochem. 61: 673-719; M. L. DePamphilis (1993), “Eukaryotic DNA replication: anatomy of an origin”, Annu. Rev. Biochem. 62: 29-63; H. Echols and M. F. Goodman (1991), “Fidelity mechanisms in DNA replication”, Annu. Rev. Biochem. 60: 477-511). The strictly controlled process of DNA replication starts in E. coli e.g. when a protein is bound (K. Geider and H. Hoffmann Berling (1981), “Proteins controlling the helical structure of DNA”, Annu. Rev. Biochem. 50: 233-260) to the highly specific initiation site thus defining the starting point of a specific RNA polymerase (primase). It synthesizes a short RNA strand (˜10 nucleotides, ‘primer’) which is complementary to one of the DNA template strands. The 3′ hydroxyl group of the terminal ribonucleotide of this RNA chain serves as ‘primer’ for the synthesis of new DNA by a DNA polymerase. DNA-untwisting proteins unwind the DNA double helix (J. C. Wang (1985), “DNA topoisomerases”, Annu. Rev. Biochem. 54: 665-697). The separated individual strands are stabilized by DNA-binding proteins as regards their conformation (J. W. Chase and K. R. Williams (1986), “Single-stranded DNA binding proteins required for DNA replication”, Annu. Rev. Biochem 55: 103-136) to enable proper functioning of the DNA polymerases (T. S. Wang (1991), “Eukaryotic DNA polymerases”, Annu. Rev. Biochem. 60: 513-552). A multienzyme complex, the holoenzyme of DNA-polymerase-III, synthesizes the majority of the new DNA. The RNA portion of the chimeric RNA-DNA molecule is then split off the DNA polymerase III. The removal of the RNA from the newly formed DNA chains creates gaps between the DNA fragments. These gaps are filled by DNA-polymerase I which can newly synthesize DNA from a single-stranded template. While one of the two newly synthesized DNA strands is synthesized continuously (5′-3′ direction, leader strand), Ogawa and Okazaki observed that a majority of the newly synthesized opposite strand (3′-5′ direction, delayed strand) was synthesized from short DNA fragments (T. Ogawa and T. Okazaki (1980), “Discontinuous DNA replication”, Annu. Rev. Biochem. 49: 421-457). Here, what is called primases initiate the onset of the DNA synthesis of the opposite strand by the synthesis of several RNA primers. When the replication proceeds, these fragments are freed from their RNA primers, the gaps are closed and covalently linked with one another to give extended daughter strands by the DNA ligase. Two chromosomes form after the termination of the replication cycle.
DNA replication is controlled in many plasmids via a replication origin which dispenses with the synthesis of the delayed strand (3′-5′ direction) and can initiate the synthesis of two continuous DNA strands bidirectionally (each in the 5′-3′ direction along the two templates). The precondition for a complete DNA replication is here the cyclic form of the nucleic acid. It ensures that at the end of the new synthesis of the complementary DNA strands the DNA polymerases return to the starting point again where now ligases guarantee the covalent linkage of the ends of the two newly synthesized daughter strands.
Smallpox viruses represent an interesting form of linear-cyclic nucleic acids: because of what is called ‘hairpin loops’ at the ends of their linear genomes, they have a cyclic molecule structure while maintaining a predominantly linear conformation (D. N. Black et al. (1986), “Genomic relationship between capripoxviruses”, Virus Res. 5: 277-292; J. J. Esposito and J. C. Knight (1985) “Orthopoxvirus DNA: a comparison of restriction profiles and maps”, Virology 143: 230-251). Covalently closed “hairpin” nucleic acids were not only found in smallpox viruses but also described for the ribosomal RNA from Tetrahymena (E. H. Blackburn and J. G. Gall (1978), “A tandemly repeated sequence at the termini of the extrachromosomal ribosomal RNA genes in Tetrahymena”, J. Mol. Biol. 120: 33-53) and the genomes of the parvoviruses (S. E. Straus et al. (1976), “Concatemers of alternating plus and minus strands are intermediates in adenovirus-associated virus DNA synthesis”, Proc. Natl. Acad. Sci. U.S.A. 73: 742-746; P. Tattersall and D. C. Ward (1976), “Rolling hairpin model for the replication of parvovirus and linear chromosomal DNA”, Nature 263: 106-109).
However, by means of the formerly known plasmids or nucleic acid constructs it is not possible to appropriately introduce nucleic acids into cells or cell organelles via the protein import route. But this is e.g. a precondition for genetically treating changes of the mitochondrial genomes of patients suffering from neuromuscular and neurodegenerative diseases or carrying out an appropriate mutagenesis in mitochondria or other cell organelles.